This invention relates to nucleic acid and amino acid sequences of a human microtubule-associated protein and to the use of these sequences in the diagnosis, treatment, and prevention of diseases associated with immune disorders and cancer.
Microtubules (MTs) are involved in many cellular functions such as mitosis, morphogenesis, motility, and intracellular organelle transport. For example, disruption of MTs by various MT-destabilizing agents results in major changes in cytoplasmic organization, including collapse of intermediate filaments and redistribution of the Golgi apparatus and endoplasmic reticulum. MTs also function in the maintenance of cell shape, movement of eukaryotic cilia and flagella, formation of the mitotic spindle, and regulation of organelle distribution and vesicle movements. Regulation of the dynamic properties of MTs is believed to play a role in many of these functions.
A group of proteins that bind MTs are defined as microtubule-associated proteins (MAPs). MAPS are involved in modulating MT dynamics and function. MAPs can be categorized into two major classes according to their primary function; motor proteins, which include the kinesin superfamily and the dynein family, and non-motor proteins, which are further divided into the high molecular weight MAPs, including MAP1A, MAP1B, MAP2, and MAP4 and the low molecular weight tau proteins. (See, e.g., Ding M. Et al. (1996) J. Biol. Chem. 271:12555-12561.) Among the identified functions of MAPs in neuronal cells, kinesin and cytoplasmic dynein are responsible for anterograde and retrograde axonal transport, respectively. Tau protein and MAP2 appear to be required for initial neurite growth. It is well known that the function of MAPs can be regulated through phosphorylation. For example, phosphorylation of MAPs can alter their affinity for MTs in vivo and affect their ability to stabilize MTs.
Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of paired helical and, straight filaments which constitute a defining neuropathological characteristic of Alzheimer""s disease and a number of other neurodegenerative disorders. Full length tau protein can assemble into Alzheimer-like filaments upon incubation with heparin, a repeating disaccharide chain. Heparin also promotes phosphorylation of tau protein by a number of kinases, prevents tau protein from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau protein and tubulin. Moreover, cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase II phosphorylate tau proteins at specific sites in vitro, some of which are also phosphorylated in PHF-tau. Hyperphosphorylation of tau protein results in its inability to bind to microtubules and is believed to precede paired helical filament assembly. Phosphorylation-independent interaction between recombinant tau protein and sulfated glycosaminoglycans leads to the formation of Alzheimer-like filaments under physiological conditions in vitro. (See e.g., Hasegawa, M. Et al. (1997) J. Biol. Chem. 272:33118-33124.)
The discovery of a new human microtubule-associated protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of diseases associated with cell proliferation.
The invention features a substantially purified polypeptide, human microtubule-associated protein (HMAP), comprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant of HMAP having at least 90% amino acid identity to the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide which is complementary to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding HMAP under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified HMAP having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist to HMAP.
The invention also provides a method for treating or preventing an immune disorders, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist to HMAP.
The invention also provides a method for detecting a polynucleotide encoding HMAP in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding HMAP in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.